This application is related to U.S. Ser. No. 09/073,538, filed May 6, 1998, which is a continuation-in-part of U.S. Ser. No. 08/846,247, filed Apr. 30, 1997, and is related to PCT application No. U.S. Ser. No. 98/08792 and U.S. provisional application Serial No. 60/076,919, filed Mar. 5, 1998, now lapsed.
The present invention provides recombinant DNA compounds and host cells containing novel polyketide synthase (PKS) genes and novel polyketides. The invention relates to the fields of chemistry, medicinal chemistry, human and veterinary medicine, molecular biology, pharmacology, agriculture, and animal husbandry.
Few molecules have captured interest in both chemotherapy and chemistry to the extent of the polyketide erythromycin and its semi-synthetic derivatives. Erythromycin and its congeners are the third most widely used class of antibiotics, with current worldwide sales exceeding US $3.5 billion. In addition, erythromycin analogs are gaining interest for their potential use in the treatment of gastrointestinal disorders (Omura, xe2x80x9cThe expanded horizon for microbial metabolitesxe2x80x94a review,xe2x80x9d Gene 115, 141-149 (1992)), inflammatory diseases (Kawasaki et al., xe2x80x9cRoxithromycin inhibits cytokine production by and neutrophil attachment to human bronchial epithelial cells in vitro,xe2x80x9d Antimicrob. Agents Chemother. 42, 1499-1502 (1998)), and as next-generation antibiotics for treatment of emerging drug-resistant strains of bacteria (Agoudiras et al., xe2x80x9cIn-vitro antibacterial activity of RU 004 (HMR 3004), a novel ketolide derivative active against respiratory pathogens,xe2x80x9d Antimicrob. Agents Chemother. 41, 2149-2158 (1997)).
The chemical challenges of erythromycin attracted the talents of R. B. Woodward and 48 colleagues who described its complete synthesis in a series of landmark publications (Woodward et al., xe2x80x9cAsymmetric total synthesis of erythromycin. 1. Synthesis of erythronolide A secoacid derivative via asymmetric induction;xe2x80x9d 2. Synthesis of an erythronolide A lactone system;xe2x80x9d and 3. Total synthesis of erythromycin,xe2x80x9d J. Am. Chem. Soc. 103, 3210-3217 (1981)), and of a cadre of medicinal chemists who prepared analogs leading to the important second generation of macrolide antibioticsxe2x80x94clarithromycin, azithromycin, and others (Chu, xe2x80x9cRecent developments in 14- and 15-membered macrolides,xe2x80x9d Exp. Opin. Invest. Drugs 4, 65-94 (1995)). Although such efforts effectively saturated the chemical modifications possible at the existing functional groups of the macrolide ring, most of the ring remained inert to chemical modification.
The modular nature of polyketide biosynthesis (Cortxc3xa9s et al., xe2x80x9cAn unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea,xe2x80x9d Nature 348, 176-178 (1990); and Donadio et al., xe2x80x9cModular organization of genes required for complex polyketide biosynthesis,xe2x80x9d Science 252, 675-679 (1991)) has facilitated genetic engineering strategies for the production of novel polyketides (McDaniel et al., xe2x80x9cRational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits,xe2x80x9d Nature 375, 549-554 (1995) and Katz, xe2x80x9cManipulation of modular polyketide synthases,xe2x80x9d Chem. Rev. 97, 2557-2576 (1997)).
The xe2x80x9cmodularxe2x80x9d PKSs are each encoded by a cluster of contiguous genes and have a linear, modular organization of similar catalytic domains that both build and modify the polyketide backbone. Each module contains a set of three domainsxe2x80x94a ketosynthase (KS), an acyltransferase (AT), and an acyl carrier protein (ACP)xe2x80x94that catalyze a 2-carbon extension of the growing polyketide chain (FIG. 1 and O""Hagan, The polyketide metabolites (E. Horwood, New York, 1991)). The choice of extender unit used by each modulexe2x80x94acetate, propionate, or other small organic acids in the form of CoA thioestersxe2x80x94is determined by the specificity of the AT domain (Oliynyk et al., xe2x80x9cA hybrid modular polyketide synthase obtained by domain swapping,xe2x80x9d Chem. and Biol. 3, 833-839 (1996); Liu et al., xe2x80x9cBiosynthesis of 2-nor-6-deoxyerythronolide B by rationally designed domain substitution,xe2x80x9d J. Am. Chem. Soc. 119, 10553-10554 (1997); and Ruan et al., xe2x80x9cAcyltransferase domain substitutions in erythromycin polyketide synthase yields novel erythromycin derivatives,xe2x80x9d J. Bacteriol. 179, 6416-6425 (1997)).
With each 2-carbon chain extension, the oxidation state of the xcex2-carbon is embedded as a ketone, hydroxyl, methenyl, or methylene group by the presence or absence of one, two, or three additional catalytic domains in the modulexe2x80x94a ketoreductase (KR), dehydratase (DH) and/or enoyl reductase (ER). In effect, the composition of catalytic domains within a module provides a xe2x80x9ccodexe2x80x9d for the structure of each 2-carbon unit, and the order of modules codes for the sequence of the 2-carbon units, together creating a linear template for the linear polyketide product. The remarkable structural diversity of polyketides is governed by the combinatorial possibilities of arranging catalytic domains within each module, the sequence and number of modules, and the post-polyketide synthesis cyclization and xe2x80x9ctailoring enzymesxe2x80x9d that accompany the PKS genes. The direct correspondence between the catalytic domains of modules in a PKS and the structure of the resulting biosynthetic product portends the possibility of modifying polyketide structure by modifying the domains of the modular PKS.
There remains a need for compounds with modifications of the chemically inert sites of polyketides such as erythromycin that can be produced by genetic engineering. Such novel macrolides could in themselves provide the basis for new pharmaceuticals or serve as scaffolds for new semi-synthetic analogs. The present invention meets this need.
The present invention provides a library of recombinant PKS genes, host cells containing those genes, and the polyketides produced by those host cells. The polyketides provided by the invention include the polyketides shown in FIG. 2, as well as the polyketides that can be prepared by any of the myriad possible combinations of the recombinant PKS genes of the invention.
The present invention also provides the glycosylated and hydroxylated forms of the polyketides of the invention that can be produced by contacting the polyketides described herein with host cells selected from the group consisting of Saccharopolyspora erythraea, Streptomyces venezuelae, S. narbonensis, S. antibioticus, S. fradiae, S. thernotolerans, and Micromonospora megalomicea. The invention also provides compounds derived from the foregoing by chemical modification, including the C-6 to C-9 hemiketals formed from the compounds of the invention having a C-6 hydroxyl group and a C-9 keto group by treatment with mild acid.
The present invention also provides novel polyketides in isolated and purified form, as well as in cultures of recombinant host cells. Particular polyketides provided include 5,6-dideoxy-10-norerythronolide B, 6-deoxy-12-norerythronolide B, 2, 10-bisnor-3-oxo-6-deoxy-10,11 -anhydroerythronolide B, and 2,4-bisnor-3-oxo-6-deoxyerythronolide B, as well as the glycosylated and hydroxylated forms thereof
The present invention also provides the polyketide compounds of the invention in the form of pharmaceutical compositions, and methods for using the same in the treatment of disease.
These and other embodiments, modes, and aspects of the invention are described in more detail in the following description, the examples, and claims set forth below.